Recovery of bacitracin



Patented Sept. 2, v1952 Haute, 1nd,, assignors to Commercial; :Solvents Corporation, .Terre Haute, -Ind. ,:ra-1corporation ofMaryland V :Noprawing. Application ocm er' rzplsre, V "SerialNo.121'-,060 I I iszolaims. (01;;167-65) This invention relates to the recovery of bacitracin. More particularly, it relates to the water extraction of bacitracin from water-immiscible aliphatic alcoholsolutions thereof.

The antibiotic bacitracin is produced by the propagation of the organism Bacillus subtilis on a nutrient mediumunder aerobic "conditions in much the same'manne'r as penicillinisjproduced. The bacitracin in the fermented-medium, however, is present only inrelativelysmallquantities,"i. e. of the order' of 50 -100 units ,per "milliliter. The separation'and recovery'of this "small amount .of material'from'ia largevolume of'medium of complex character presents numerous difficulties. 7

The prioriproce'ssesare'illustratedby the disclosure in' U. S. Patent'No."457,887, granted January4,'1949,'t'o 'J. '1. 'Goorley. 'According'to the disclosure of this vpatent,bacitracin is first separated from the fermented medium in which it is produced by adsorption on an adsorbent such as activated .aluminaor activated carbon. After separating the adsorbed material from the fermentation medium .the bacitracin is eluted from the adsorbent by treatment with a mixture of a dilute aqueous solution .of anacidsuch as hydrochloric acid and anorganic. solvent that'is inert with respect to bacitracin, isuch as butanol orother water insoluble aliphatic alcohol. After neutralization of the acidsolutionthe butanolor other similar solution is next treated with an adsorbent to remove impuritieaifollowed by'extraction of the liquid with water and-an immiscible organic solvent, such as chloroform, which decreases the solubility of'bacitra'cin in butanol so that the bacitracin goes into the water phase which may then .belconcentrated or carried to dryness.

According to an alternate procedure, the bacitracin may beextracted directly from the fermented medium at a pH close to 7.0 with a solvent such as butanol-instead of by adsorption followed by elution as described above. This step is followed by the transfer of bacitracin from butanol to water. It consists of first mixing the butanol with ether and water. -Sinceether is more soluble in butanol than in water, itgoes .into solution in the butanol driving the -bacitracin into the water layer. This step isrepeated several times ending up withthe bacitracin in the water layer from which the bacitracin can be recovered by the use .of;suitable tdryingsmethods.

.The above and .all ",other'previously suggested processes :for the recovery rand :purlfication of bacitracin and particularly .the vstep :of :transbacitracin or byany other suitable means.

ferring bacitracinfromi butanol into water have been-'subjectito numerous disadvantages because of the low yields of relatively-impure bacitracin which are obtained. We have now discovered that the overall yields or bacitracin can be very mate'rially increased and the recovery 'an'd puri- 'fica'tion procedure shortene'd and simplified by following our improved process for-thetransfer of the bacitracin from the"butanol-solution -into water from which the ba'citracin is subsequently :rec'overed i by drying or "other i suitable methods. 'By our improved process we-are'abl to transfer substantially an of the bacitracinin the butanol solution 1 into a water. I

In carrying out-our improved process for the a recovery and purification pf "bacitracin we first produce '-a""butano'1, or other water-immiscible aliphatic alcohol, -=solution of "bacitracin either -by direct extractionat pHclose-td'L-O, preferably MI 755 of fermented -me'dium containing This "butanol solution containing -b'acitracin -is next mixed"W-itliwater and the pH of the resulting solution {adjusted within particular limits "with an-acid selected from the group consisting of phosphoric, pyrophosphoric, sulfuric, 'and citric acid. This mixture separates into "analcohol layer and a water-layer -containing'the"bacitracin. The ratio 'of the concentration "of bacitracin left in the butanol in units permlpto'the'concen- :tration of bacitracin-in waterto which it-has been extracted in units perrml. is knownas the distribution co'efiicient. This coeiiicient is indicative-of the success of the-extractionprocess and since the extraction isfrom butanoltowater, it is desirable to obtain distribution coefficients as low'as possible. I

In the single -extra ction operation in our improved process we have obtained-distribution coefficients ranging "from 0.02 to 0:4, depending upon the additiveused to regulate the "pH-of the solution and=the conditions under which the extraction .wa's carried out as: compared to the distribution coefficient of approximately 1 when hydrochloric acid is used under the same conditi'ons :to :adjust .the 'p'H and even after :as' many as six successive extraction contacts of new volumes ofzwater with-the same-butanol solution "the distribution 'co'efiicient reached an average IOfiOIllY approximately 0i25 for the-overall-extrac- :tion when hydrochloric acid was used. This com- .pares with a distributioncoefficient as low- 'as i0.02' vvith no more th'an iextraction-contacts in hydrochloric acid a sing'lextraction transferred of other solvents required in the prior art processes. The recovery of even a relatively small additional amount of expensive bacitracin ob- In the extraction of bacitracin from the fermented medium the solvent customarily used to effect extraction is butanol. The lower aliphatic alcohols are not operative due to the fact that they are completely miscible with water and accordingly the extraction *of bacitracin from the alcohol into water cannot be accomplished. The

water-immiscible homologs of butanol as for example the amyl alcohols can also be satisfactorily employed in our process. Similarly, aliphatic alcohols with molecular weights higher than that of amyl alcohol are operative to a certain degree viously amounts to a very important advantage in the use of our process. These together with the greatly reduced number of operations required to give a higher yield of the desired prod-- not have resulted in very important savings.

In carrying out our improved process for the extraction of bacitracin from butanol or other water-immiscible aliphatic alcohol, with water we 7 have discovered that the selection of the acid used in adjusting the pH of the mixture is of extreme importance. The use of phosphoric, pyrophosphoric, sulfuric and citric acids gives yields which greatly exceed those when other acids are used. Of the acids which we found to give good results we prefer to use phosphoric since with it the distribution coefficient obtained is less than 0.1 and the optimum pH can be reached without the use of large quantities of acid. The distribution coemcient obtained with the other named acids increases progressively up to approximately 0.3 with citric acid for a single extraction. But, with citric acid, a yield of approximately 72% bacitracin was obtained in a single extraction.

We have discovered that in order to obtain satisfactory results in our improved process the pH of the aqueous layer must be maintained at a value below approximately 4.0, the distribution coeflicient decreasing as the pH is lowered to 2.0 and increasing slightly as the pH is lowered below 2.0. Unfortunately, however, bacitracin becomes unstable at low pHs, 10% of the activity being lost at a pH of 1.6 when allowed to stand four hours at 27 C. and 33% at the end of 24 hours. Unless, therefore, the extraction is completed in a relatively short time interval and the pH of the solution promptly raised it is impractical to carry out the extraction at a pH lower than 2.0. We have accordingly, therefore, found it most practical to operate our process at a pH of approximately 2.0 since bacitracin destruction is low and the distribution coefficient at a minimum at this point. Below this pH the rate of destruction rapidly increases and above it and below it the distribution coefiicient increases.

We have found that the distribution coefiicient also is dependent on still another factor, namely the volume ratio of water-immiscible alcohol such as butanol to water. For example, as the ratio of alcohol to water decreases the distribution coefficient also decreases. This relation is inconsistent with one of the objects of the extraction, that is, volume reduction. However, if more than one extraction of the butanol is to be made considerations of volume reduction can largely be disregarded in carrying out the first extraction. We have found that volume ratios up to volumes of solvent to 1 volume of water are operative in our process, the optimum results, however, being obtained when using a 1:1 ratio. Where, however, a substantial reduction in volume is required it is sometimes advisable to use higher ratios even though a somewhat higher distribution coefficient is thus obtained. i

but bacitracin becomes more insoluble as the molecular weight of the alcohol increases and alcohols above amyl alcohol are accordingly not generally usedvin the extraction of bacitracin from the fermented medium.

In carrying out our invention we prefer to employ the following procedure; however, we do not intend to limit ourselves to it, it being understood that the ratios of solvent to Water can be varied as desired in accordance with the disclosure made above: to 1 part of a water-immiscible alcoholic extract, such as the butanol or amyl alcohol extract of bacitracin from a fermentation beer, 1 part of water is added. The mixture is agitated and the pH adjusted to 2.0, preferably with phosphoric acid. Agitation is continued for approximately 15 minutes and the mixture is then allowed to form an aqueous layer-and a water-immiscible alcohol layer. The layers are separated and if desired the process is repeated by reextr'acting the water-immiscible alcohol layer with an 'equal volume of water. .The composite of the water layers containing the bacitracin is then treated in any manner desired .for the recovery. of the bacitracin, as for example by dryingor by an extraction followed by removal of'the solvent.

The following examples are givenfto illustrate my invention and are not to be construed as limiting it to the exact conditions prescribed:

i EXAIVIPLE I u./m1. in butanol u./ml. in H O being 0.02.

EXAMPLE 11 One volume (ml) of a butanol extract of bacitracin was mixed with 1 volume. (100 ml.) of water. Themixture was agitated andthe pH adjusted to 3.0 by the addition of phosphoric acid. Agitation was continued for an additional 15 minutes and the mixture then allowed-to separate into layers. Analysis of the-layers showed that 94% of the bacitracin had been transferred to the water layer.

. H U nxAi/LP LE I II Ten volumes (4000 ml): of a butanol extract of bacitracinlcontaining' u./ml. from a bacitracin. fermentationrbeer was mixed with 1 volume (400-.m1.)- of water. j The mixture was -5. agitated and the pH adjusted to 2.0 with phosphoric acid. Agitation was-then continued for an additional 15 minutes and the mixture then permitted to form into layers and the latter separated and assayed. The butanol Iayer Was reextracted several times with-fresh 400 ml. portion's-of water, thepH' being adjusted to 2.0 with phosphoric acid for each extraction. The-results obtained after each extraction are shown To one volume (100 ml.) of butanol and 1 volume (100 ml.) of water was added 2.8 g. of bacitracin. The mixture was agitated and the pH adjusted to 2.0 by the addition of pyrophosphoric acid. Agitation was then continued for an additional 15 minutes and the mixture permitted to form into layers which were then separated and assayed. The water layer, 88 ml., contained 1400 u./ml. ml., contained '70 u./ml. The distribution coefficient for the extraction was 0.05.

EXAMPLE V One volume (100 ml.) of a butanol extract of bacitracin was mixed with 1 volume (100 ml.) of water. After agitating, the pH of the mixture was adjusted to 2.0 with citric acid. Agitation was thereafter continued for an additional 15 minutes and the mixture then permitted to form into layers which were separated and assayed. The water layer, 88 ml., contained 758 u./ml. The butanol layer, 111 ml., assayed 226 u./ml.

The butanol layer, 105

The distribution coefiicient for the extraction was 0.3.

EXAMPLE 'VI for each extraction. The results of each extraction are shown in Table II.

Table II Distribution Coeflicient Contact Number A seriesof experiments was carried out to. show the effect of variation of the volume ratio of butanol to water. In each experiment the pH was adjusted to 2.0" with phosphoric acid, the procedure used being that of Example I. The

jresults" a e shown in Table III.

Table... III. -j i:

Distri- Volume ratio of butanol to water 6 33;}

- I cient EXAMPLE VIII A series of experiments was carried out to show the efiect of variation of the pH. In each experiment one volume ml.) of a butanol extract of bacitracin from a bacitracin fermentation beer was mixed with 1 volume (100 m1.) of water. The mixture was agitated and the pI-I adjusted by the addition of phosphoric acid. Agitation was continued for an additional 15 minutes and the mixture was allowed to separate into layers which were then analyzed. The results are shown in Table IV.

Table IV 11 1011 PH Oceflicient Now having disclosed our invention what we claim is: I

1. In a process for the recovery of bacitracin, the step which comprises extracting bacitracin from a solvent selected from the group consisting of butyl alcohol and amyl alcohol with an aqueous solution of an acid selected from the group consisting of phosphoric, pyrophosphoric, sulfuric and citric acid, said extraction mixture having a pH lessthan 4.0. v

2. In a process for the recovery of bacitracin, the step which comprises extracting bacitracin from a solvent selected from the group consisting of butyl alcohol and amyl alcohol with from 0.1 to 1 volume of water per volume of said solvent,

' the acidity of said extraction mixture being adtion is eifected by repeated extractions each employing at least 0.1 volume of acidified water per volume of alcohol.

MURRAY SENKUS. PETER c. MARKUNAS'.

(References on following page) 7 .8 REFERENCES CITED "QTHERLREFERENCESV 7 ...Ank.eri1.on Bacitracin: in 3... Back, Feb. 1943, volume 55;;page 252.

The following references are of record in the v GoOrIeyifif-Some Chemical and Physical Propfile of this patent::

UNITED' STATES PATENTS V v. 5 erties of Baeitracimfipages 11:0 3, paper presented n j at Conference on,Antibioties Research in Wash- Number Name l Date; ington, DEC, on. January Ell-February 1,1947,

2,457,887 Goorley Jan. 4, 1949 auspices National Inst. Health, Bethesda, Mary- 2,476,085 Dimick July 12, 1949 land. 

1. IN A PROCESS FOR THE RECOVERY OF BACITRACIN, THE STEP WHICH COMPRISES EXTRACTING BACITRACIN FROM A SOLVENT SELECTED FROM THE GROUP CONSISTING OF BUTYL ALCOHOL AND AMYL ALCOHOL WITH AN AQUEOUS SOLUTION OF AN ACID SELECTED FROM THE GROUP CONSISTING OF PHOSPHORIC, PYROPHOSPHORIC, SULFURIC AND CITRIC ACID, SAID EXTRACTION MIXTURE HAVING A PH LESS THAN 4.0. 